Project Summary: Despite impressive therapeutic advances in the treatment of CLL over the past decade, relapsed tumors that are often resistant to known therapies remain a problem. Understanding resistance and developing new treatment strategies will be necessary to turn CLL into a curable disease. With few notable exceptions, responses to chemotherapy typically occur through apoptosis, a form of programmed cell death. Our goal is to identify drugs that sensitize CLL cells to apoptosis, and to identify combinations of drugs that would prevent relapse. While chemical screening of conventional cytotoxicity is an attractive strategy to meet this goal, such an approach is impeded by the inability to reliably culture CLL cells for longer than 48 hours ex vivo. This ultimately reflects a technological gap in our ability to chemically perturb CLL ex vivo, and measure functional and clinically relevant phenotypes. Here, we will use a novel chemical screening approach called dynamic BH3 profiling (DBP), which enables functional measurements of chemically induced apoptotic changes, and requires only 6-24 hours of ex vivo culture, thereby maximizing molecular fidelity and tumor cell viability. Using a panel of over 2000 drugs, not only will we identify novel molecules that sensitize CLL cells for apoptosis, but we will also determine chemical apoptotic sensitivities that are lost or gained on relapse for individual patients. Drugs that uniquely sensitize only relapsed tumors present exciting opportunities for combination chemotherapy. Finally to understand mechanisms of apoptotic vulnerabilities in pre- and post-treatment samples, we will correlate our measurements of functional apoptotic responses with the large molecular datasets in Project 1 and 2 for at least 104 CLL patients. In sum, the successful identification of apoptotic sensitizing drugs in CLL, will not only advance our molecular understanding of CLL, but could result in truly novel therapeutic options.